Platelet aggregation, the process by which platelets adhere to other platelets at sites of vascular injury.
Platelet aggregation, the process by which platelets adhere to other platelets at sites of vascular injury, has long been recognised to be critical for haemostatic plug formation and thrombosis. Haematology laboratories perform platelet aggregation testing to diagnose specific bleeding disorders when a patient sample gives abnormal results to routine coagulation assays.
The response of the patients’ platelets in relation to different agonists, and different agonist concentrations, will show if the platelets have any qualitative or quantitative defects linked to specific disease states.
Disease states such as Glanzmann’s Thrombasthenia, Bernard-Soulier Syndrome and Storage Pool Disorders are diagnosed through interpretation of the response of patients’ plasma to a panel of agonists that reproduce platelet aggregation in vitro.
Platelet aggregation is also used in the diagnosis and typing of von Willebrands Disease, the most common hereditary bleeding disorder, using different concentrations of Ristocetin to type the disease state.
This is subsequently performed after an abnormal result is obtained from a von Willebrands Activity assay (also called Ristocetin Cofactor Activity assay) screening test. The gold standard methodology is the Born method utilising a light transmission platelet aggregometer.
Interpretation of the aggregation response is determined by evaluating maximum aggregation, final aggregation, lag phase, primary slope, secondary slope and time to maximum aggregation values for each agonist tested.
Platelets are required to adhere to the subendothelium of a vascular injury in order to initiate and perpetuate platelet plug formation. There are three main stages in platelet plug formation:
Platelets are produced by fragmentation of megakaryocytes with around 4000 platelets generated per cell. A normal circulating platelet count is between 150 – 400 x 109 / L, however only two-thirds of total platelets are circulating with one-third stored in spleen. 10% of the platelet pool is replaced every 24 hours.
They are discoid in shape, around 2 - 4 microns in size (Red blood cells are 7.5 microns) and composed of anucleated membrane bound cytoplasm housing a complex structure of microtubules, microfilaments and organelles/granules containing lysosomes, hydrolytic enzymes, peroxisomes, catalase, dense bodies, ADP, Ca2+, Serotonin, alpha granules, Fibrinogen, vWF, Factor V, PDGF and PF-4.
Bernard-Soulier Syndrome (BSS)
BSS is a relatively severe bleeding disorder caused by a genetic defect in GP Ib or GP IX, which is autosomal recessive inheritance. Bleeding time is prolonged with both Thrombocytopenia and giant platelets. Platelets in patients with BSS do not agglutinate in response to Ristocetin (GP Ib is unable to bind vWF).
von Willebrand Disease (vWD)
vWD is the most common hereditary coagulation abnormality described in humans, although it can also be acquired as a result of other medical conditions. It arises from a qualitative or quantitative deficiency of von Willebrand Factor (vWF), which is a multimeric protein that is required for platelet adhesion. vWD disease is characterized by a reduced or abnormal function of vWF and can be subdivided into different types and sub-types:
Glanzmann’s Thrombasthenia (GT)
GT is an autosomal recessive disease caused by lack of expression / qualitative defects in GP IIb / IIIa. This causes a severe impairment or lack of platelet aggregation by all agonists.
Storage Pool Disorder (SPD)
SPD is characterized by defects in the alpha or dense granules in platelets, particularly a lack of granular non-metabolic ADP. Patients with ADP deficient "Storage Pool Disease" present a prolonged bleeding time due to impaired aggregation response to fibrillar Collagen.
Aspirin Like Disorder or Aspirin Ingestion
Aspirin inhibits the release of ADP from platelet storage pool, negating activity and preventing any platelet diagnostic testing until the platelet pool recovers. Disease states like Reye’s syndrome can mimic the diagnostic results of patients who have ingested Aspirin
Arachidonic Acid (AA)
AA is a substrate for synthesis of Thromboxane A2 (TxA2). Aspirin permanently inactivates COX 1 & 2 preventing Arachidonic Acid conversion to Thromboxane A2. This Aspirin effect is evident for the lifespan of the platelets 7 – 10 days. AA is the initial screening test for Aspirin ingestion.
Adenosine Diphosphate (ADP)
ADP causes platelets to swell and encourages platelet membranes to adhere to each other. Low concentrations of ADP cause only primary, incomplete, reversible aggregation. Higher concentrations create biphasic aggregation and irreversible secondary aggregation caused by TxA2 formation. High concentration ADP causes both phases to fuse into a single phase.
Collagen binds directly to membrane protein Ia, initiating aggregation. The characteristic response is a prolonged lag phase as TxA2 formation and secretion of granule contents is required prior to aggregation.
Epinephrine inhibits cAMP formation & stimulates TxA2 formation. TxA2 inhibiting drugs will not allow aggregation in response to Epinephrine.
Ristocetin facilitates binding of vWF to GP Ib / IX / V. It produces a dose response testing for sensitivity. A normal patient requires both functional vWF and normal GP Ib / IX / V. A higher than normal aggregation response is seen for vWD Type 2B.
Platelet agonists are available from Helena Biosciences in high stock concentrations to allow for high and low dose dilutions recommended by CLSI guideline H58-A Platelet Function Testing by Aggregometry; Approved Guideline. They are suitable for use with any platelet aggregometer, including the AggRAM Platelet Aggregometer which utilises the gold standard Born methodology (light transmission) and is also available from Helena Biosciences Europe:
The AggRAM offers fully customisable platelet aggregation and ristocetin cofactor testing by combining a high performance analyser with powerful software that allows up to 21 patient results to be overlayed and compared simultaneously.